mmg_233_2013_genetics_genomicswikiaorg-20200214-history
Archaea predominate among ammonia-oxidizing prokaryotes in soils
Ammonium oxidation is an important part of the nitrification process that results in the formation of nitrate through microbial activity. In this study, it is shown the archaeal ammonia oxidizers are more abundant in the soils than bacterial ammonia oxidizers. To do this, the gene encoding ammonia monooxygenase is investigated for its abundance. Using reverse transcription quantitative PCR studies and complementary DNA analysis using novel cloning-independent pyrosequencing technology showed that crenarchaeota may be the most abundant ammonia oxidizing organisms in the soil ecosystems. Materials/Methods The extraction and preparation of nucleic acids were performed from a modified version of the method described in ref. 25. The cDNA was prepared as described previously. DNA concentration was measured by incubating dilutions of extracted DNA with the fluorescent dye SYBRGreenI. Real time PCR was also performed with 5 ng of DNA pooled from each soil sample. This was used to quantify the copy numbers of archaeal amoA genes and transcripts. This procedure was also done for amoA from bacteria using bacterial amoA specific primers. GDGTs were extracted from the soil with a polar solvant and cleaned by Al2O3 solid phase extraction and filtration through 0.45um polytetrafluoroethylene filters. The fractions were analyzed by liquid chromatography atmospheric pressure chemical ionization mass spectrometry on a cyanopropyl column and protonated molecular ions were recorded in selected ion monitoring. The cDNA was prepared by RNA isolation from RUD soil and subjected to reverse transcription with random hexanucleotides and second strand synthesis. In this study, two biomarkers were focused on to quantify the ammonia oxidizing crenarchaeota in terrestrial ecosystems, the amoA gene, and tetraether lipids diagnostic for crenarchaeota. Results/Discussion It has been recently found that the genes encoding ammonia monooxygenase was found to affiliated with the phylum of Crenarchaeota. It was found that archael amoA genes were high in contrast to bacterial amoA genes that varied considerably over three orders of magnitude. The archaeal amoA genes dominated over bacterial amoA genes in all soils that were sampled. The distribution of ammonium oxidizing archae (AOA) and ammonium oxidizing bacteria (AOB) were analyzed at different depths. Agricultural soil that has been treated with different amounts and qualities of fertilizers and unfertilized soil were investigated and it was found that bacterial amoA genes declined significantly with depth in the unfertilized while archael amoA genes stayed high. The relative abundance of AOA with respect to the total microbiota varied mostly between 1% and 5% while the highest fraction of AOB in soil microbiota was only 0.23%. Isoprenoid hlycerol dialkyl glycerol tetraethers (GDGTs) are membrane lipids of archaea and was used to study their presence and distribution. The amount of archaea derived isoprenoidal GDGT in ten of the the soils were investigated and the values ranged from 0.04 to 3.24 ug per g of soil and a significant fraction was crenarchaeol which shows that it occurs in considerable amounts in the soil ecosystems. The data provides evidence for high abundance of AOA in soils and suggests that they represent the most abundant ammonia oxidizing organisms in soil ecosystems. Reference 1. Archaea predominate among ammonia-oxidizing prokaryotes in soils (PDF Download)